Quantitative detection of platelet aggregates in whole blood without fixation.

Platelet counting detects lesser degrees of platelet aggregation than conventional aggregometry. In order to prevent progressive platelet aggregation or disaggregation after sampling it is customary to fix blood samples. However fixation may introduce other artefacts. We first compared stability of platelet counts in EDTA-, citrate- and r-hirudin-anticoagulated blood from healthy volunteers. Second, the stability of platelet counts in unfixed EDTA- and hirudin-anticoagulated blood was compared with glutaraldehyde-fixed blood in the same anticoagulants. Third, the effect of in vivo heparin administration on platelet counts in EDTA- and hirudin-anticoagulated blood was studied. Platelet counts within 2 h of collection were significantly higher in EDTA- than in hirudin- or citrate-anticoagulated blood (P = 0.002 vs. hirudin and P = 0.001 vs. citrate). Twenty-four hour counts in hirudin and EDTA were unchanged (P = 0.3 and P = 0.2, respectively, vs. earlier counts). Counts in citrate increased significantly (P = 0.007; n = 10). Platelet counts in fixed blood did not differ significantly from those in unfixed blood. Heparin administered for cardiopulmonary bypass reduced platelet counts in hirudin-anticoagulated blood from (mean +/- 1 standard deviation) 180 +/- 45 to 162 +/- 30 x 10(9) l-1 (P = 0.01; n = 14), without significantly lowering counts with EDTA-anticoagulation, consistent with increased platelet aggregation. Hirudin and EDTA provided stable platelet counts, suggesting that fixation is unnecessary.

Heparin-induced platelet dysfunction and cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass is associated with impaired platelet macroaggregation. Heparin contributes to platelet dysfunction before extracorporeal circulation. In vitro heparinization of whole blood does not impair macroaggregation. Heparin releases several endothelial proteins; thus heparin may inhibit macroaggregation indirectly. METHODS: Patients undergoing operations using cardiopulmonary bypass and ABO blood group compatible volunteers were studied. Whole blood impedance aggregometry assessed macroaggregation in response to collagen (0.6 microg ml(-1)) in blood diluted either with normal saline or with platelet poor plasma, obtained from patients at different stages of cardiopulmonary bypass. RESULTS: Before heparinization, blood diluted with its own platelet poor plasma recorded an impedance change of 13.0 (4.7 to 15.6) Ohms. Platelet poor plasma obtained after heparinization or during extracorporeal circulation reduced this response to 3.7 (1.1 to 8.4) and 2.0 (1.1 to 3.3) Ohms, respectively (both p < 0.0001 versus pre-heparin; n = 13). Macroaggregation in blood from volunteers was similarly inhibited by patients' platelet poor plasma (n = 30). The macroaggregatory response in blood sampled after heparinization for cardiopulmonary bypass, decreased gradually from 11.4 (8.2 to 15.9) Ohms immediately after sampling to 1.7 (1.4 to 4.1) Ohms 2 hours later (p < 0.0001; n = 11). CONCLUSIONS: In vivo heparinization induces plasma changes that inhibit platelet macroaggregation. This is an indirect, delayed inhibition that is transferable in vitro to normal platelets.

Platelet 5-hydroxytryptamine is decreased in a preliminary group of depressed patients receiving the 5-hydroxytryptamine re-uptake inhibiting drug fluoxetine.

1. In view of the importance of 5-hydroxytryptamine in coronary thrombosis, we wanted to know whether a potentially protective decrease in platelet 5-hydroxytryptamine could be achieved by treatment with an inhibitor of 5-hydroxytryptamine uptake, fluoxetine. 2. We studied 15 patients treated for psychiatric indications with fluoxetine, and compared the findings with those obtained with blood from 18 patients treated with amitriptyline and 13 controls previously treated for affective disorders. 3. Platelet-rich plasma 5-hydroxytryptamine levels were significantly decreased in the fluoxetine group (P < 0.005) but not in the amitriptyline group compared with the control group. 4. Collagen-induced aggregation in whole blood anticoagulated with hirudin was measured by sequential single platelet counting. The contribution of 5-hydroxytryptamine was assessed from the effect of adding the 5-hydroxytryptamine specific antagonist ICI 170809. This contribution was significantly decreased in the fluoxetine group but not in the amitriptyline group compared with the control group. 5. It is concluded that platelet 5-hydroxytryptamine is indeed decreased by fluoxetine, and we would predict a protective effect of fluoxetine against coronary thrombosis.

Impaired platelet aggregation after cardiopulmonary bypass in man: enhancement of collagen-induced aggregation in whole blood and plasma by adrenaline ex vivo.

1. We tested the effect of intravenous adrenaline at 0.55-1.10 nmol min-1 kg-1 (for 3-8 min, at 7-10 min post bypass; n = 7) on both microaggregation in hirudinized whole blood, using platelet counting, and macroaggregation in platelet-rich plasma, using optical aggregometry. Control (n = 12) blood samples were taken before and at 10 and 20 min after bypass. 2. Post-bypass plasma adrenaline levels (nmol/l) increased slightly in controls (1.0 versus 0.7 at 10 min, medians; P = 0.05) and markedly with adrenaline infusion (36 versus 0.5 before infusion, P = 0.02). Microaggregation (percentage decrease in single platelets) in stirred blood, reflecting largely ADP-dependent 'spontaneous' aggregation, was not influenced by adrenaline infusion. In contrast, collagen (0.2 microgram/ml)-induced microaggregation in blood was enhanced by adrenaline (92% versus 41%, P = 0.02), with no change in controls (60% versus 53%, P = 0.61). 3. In controls, collagen (0.6 microgram/ml)-induced macroaggregation in platelet-rich plasma (extent of increase in light transmission, cm) was impaired at 10 min post bypass (5.3 versus 12.1 before bypass, P = 0.01), but was enhanced by adrenaline (7.0 versus 3.6 before infusion, P = 0.02). Platelet counts (x 10(9)/l) were decreased postbypass (155 versus 220, P = 0.02) and were not influenced by adrenaline infusion (167, P = 0.93).(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous platelet aggregation in whole blood is increased in non-insulin-dependent diabetes mellitus and in female but not male patients with primary dyslipidemia.

Increased platelet aggregability has been shown in hypercholesterolemia, and stirring-induced spontaneous aggregation in whole blood is increased in insulin-dependent diabetes mellitus (DM). We have determined spontaneous aggregation in citrated (10 mM) whole blood, from 27 primary dyslipidemic patients (DYS; 14F, 13M), 16 male non-insulin-dependent DM (NIDDM) patients, and 17 normolipidemic controls (N; 6F, 11M), using platelet counting to quantify aggregation. Spontaneous aggregation was significantly higher, both in the female DYS group (median 30% [interquartile range 25,50], P < 0.005) and the NIDDM group (33% [25,41], P < 0.005), than in the N group (17% [12,27]), but did not differ significantly in the male DYS group (23% [10,33]). Similar results were obtained in the presence of indomethacin (25 mumol/l) to prevent artefactual thromboxane (TX) A2 formation, indicating that increased spontaneous aggregation was TXA2-independent. Interestingly, increased spontaneous aggregation appeared to be independent of serum cholesterol and triglyceride concentrations, as well as age and sex per se. We conclude that spontaneous platelet aggregation was increased both in female primary dyslipidemic patients and NIDDM patients, but not in male DYS patients. The clinical significance of increased spontaneous platelet aggregability is that it may favour shear-induced aggregation which may occur at critical arterial stenoses in vivo leading to thrombus formation.

The DP-receptor Dependence of the Inhibitory Effects of PGD, on Collagen-induced Platelet Aggregation and TXA, Synthesis in Human Whole Blood in Vitro.

The DP receptor-dependence of inhibition of platelet aggregation (PA), degranulation, and TXA(2) synthesis, by PGD(2), in human whole blood, has not been established since selective antagonists have only recently been developed. Accordingly, the effects of PGD(2) (30 nM), were determined using the DP receptor antagonists AH6809 (50 µM) and 868C84 (0.5 µM), and results were compared with those obtained using the stable and DP receptor-specific agonist 572C85 (30 nM). With collagen at 0.3 µg/ml, PGD(2) markedly inhibited PA (6 vs 91% PA, p >0.03, n = 12) and both AH6809 and 868C84 alone also inhibited PA but less markedly (62 and 63% PA, respectively) and both antagonists largely prevented the antiaggregatory action of PGD(2) (57 and 52% PA, respectively). PGD(2) also markedly inhibited TXB(2), formation (reflecting inhibition of TXA(2) synthesis) (19 vs 48 nM TXB(2), p>0.03). AH6809 and 868C84 alone had little effect (both 45 nM) but both antagonists significantly reduced the inhibitory effect of PGD(2) on TXB(2) formation (35 and 33 nM, respectively, p> 0.03 vs PGD(2) alone). PGD(2) also inhibited ß-thromboglobulin release, but only to a similar extent as with AH6809 and 868C84 alone. With collagen at 3.0 µg/ml, PGD(2) again inhibited PA (60 vs 96% PA, p >0.03), AH6809 and 868C84 alone had no effect on PA (98 and 96% respectively) but effectively abolished the antiaggregatory effect of PGD(2). PGD(2) also inhibited TXB(2) formation (194 vs 339 nM, p > 0.03) and this effect of PGD(2) was effectively abolished both by AH6809 and 868C84 (313 and 308 nM, respectively). Results obtained with 572C85 largely confirmed those obtained with PGD(2), and with collagen at 0.3 µg/ml, 868C84 effectively abolished inhibition of both PA and TXB(2) formation by 572C85. Thus, DP receptor-dependent inhibition of both aggregation and TXA(2) synthesis both by PGD(2) and the more selective DP receptor agonist 572C85, was established using the DP receptor antagonists AH6809 and 868C84. Results obtained for ß-thromboglobulin release were inconclusive since both AH6809 and 868C84 inhibited release to a similar extent as did PGD(2), indicating that a limited effect either on aggregation or TXB(2) formation does not preclude a greater effect on degranulation.

Original article: the influence of aspirin on the proaggregatory action of adrenaline after cardiopulmonary bypass in man.

We recently showed that collagen-induced platelet aggregation (PA) is enhanced by adrenaline ex vivo following cardiopulmonary bypass in man. Collagen-induced PA is impaired following bypass and this may contribute to postoperative blood loss, and aspirin treatment may further aggravate bleeding. To determine the influence of aspirin on the proaggregatory action of adrenaline following bypass, we assessed the effect of aspirin on collagen-induced PA before and after bypass, and following adrenaline infusion after bypass. Using optical aggregometry and hirudinised platelet rich plasma, in non-aspirin-treated controls (n=6), collagen (1.0 pg/ml) induced PA (median amplitude; cm) was maximal before bypass (13.7) and impaired at 10 min after bypass (11.5, P=0.03). PA was inhibited by aspirin (1 mM) before bypass (7.2 vs 13.7, P=0.03), and following bypass in aspirin-treated controls (3.3 vs 11.5, P=0.03). In aspirin-treated patients given adrenaline after bypass (n = 6), PA before adrenaline was impaired to a similar extent as in aspirin-treated controls (2.4 vs 3.3), but was enhanced following intravenous adrenaline infusion (0.55-1.1 nmol kg(-1) min(-1)) for 3-10 min (5.2 vs 2.4, P=0.03), with no change in controls at 20 min post-bypass (4.1 vs 3.3). This study indicates that at normocalcaemia, adrenaline enhanced collagen-induced PA in aspirin-treated patients, despite impairment of PA, but only at a high concentration of collagen. These findings indicate that aspirin-treatment may further impair collagen-induced PA following bypass, by limiting the proaggregatory effect of adrenaline.

Macroaggregation of platelets in plasma, as distinct from microaggregation in whole blood (and plasma), as determined using optical aggregometry and platelet counting respectively, is specifically impaired following cardiopulmonary bypass in man.

We determined changes in platelet aggregability following cardiopulmonary bypass, using optical aggregometry to assess macroaggregation in platelet-rich plasma (PRP), and platelet counting to assess microaggregation both in whole blood and PRP. Hirudin was used as the anticoagulant to maintain normocalcaemia. Microaggregation (%, median and interquartile range) in blood stirred with collagen (0.6 micrograms/ml) was only marginally impaired following bypass (91 [88, 93] at 10 min postbypass v 95 (92, 96] prebypass; n = 22), whereas macroaggregation (amplitude of response; cm) in PRP stirred with collagen (1.0 micrograms/ml) was markedly impaired (9.5 [8.0, 10.8], n = 41 v 13.4 [12.7, 14.3], n = 10; p < 0.0001). However, in PRP, despite impairment of macroaggregation (9.1 [8.5, 10.1], n = 12), microaggregation was near-maximal (93 [91, 94]), as in whole blood stirred with collagen. In contrast, in aspirin-treated patients (n = 14), both collagen-induced microaggregation in whole blood (49 [47, 52]) and macroaggregation in PRP (5.1 [3.8, 6.6]) were more markedly impaired, compared with control (both p < 0.001). Similarly, in PRP, macroaggregation with ristocetin (1.5 mg/ml) was also impaired following bypass (9.4 [7.2, 10.7], n = 38 v 12.4 [10.0, 13.4]; p < 0.0002, n = 20), but as found with collagen, despite impairment of macroaggregation (7.2 [3.5, 10.9], n = 12), microaggregation was again near-maximal (96 [93, 97]). The response to ristocetin was more markedly impared after bypass in succinylated gelatin (Gelofusine) treated patients (5.6 [2.8, 8.6], n = 17; p < 0.005 v control), whereas the response to collagen was little different (9.3 v 9.5).(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of PGD(2) Contributes to the Anti-aggregatory Efficacy of ZD1542, a Thromboxane A(2) Synthase Inhibitor and TP Receptor Antagonist, in Human Whole Blood.

The aim of this study was to determine the contribution of increased PGD(2) formation to the anti-aggregatory action of a thromboxane A(2) (TXA(2)) synthase inhibitor and TP receptor antagonist (ZD1542) against collagen-induced human platelet aggregation in citrated whole blood using the prostaglandin (PG) DP receptor antagonists AH6809 and 868C84. ZD1542 (1 µM) was markedly more effective than aspirin (1 mM) against collagen-induced aggregation (73 vs 52% inhibition respectively), and formation of both PGI(2) and PGD(2) was evident in the presence of ZD1542. Added PGD(2) (30 nM) inhibited aggregation to a similar extent as did ZD1542 (68% inhibition) and the antiaggregatory action of PGD(2) was abolished, both by AH6809 (50 µM) and by 868C84 (0.5 µM). AH6809 significantly reduced the antiaggregatory efficacy of ZD1542, but did not modify the efficacy of aspirin. In contrast, 868C84 had little effect on the antiaggregatory efficacy of ZD1542 whereas 868C84 in combination with aspirin inhibited platelet aggregation more markedly than did aspirin alone. These findings indicate that formation of PGD(2) contributes to the antiaggregatory action of ZD1542 against collagen-induced human platelet aggregation. However, results obtained with the DP receptor antagonist 868C84 were inconclusive since 868C84 has a limited inhibitory effect against collagen-induced human platelet aggregation which is independent of eicosanoid formation.

Collagen induced human platelet aggregation: serotonin receptor antagonism retards aggregate growth in vitro.

OBJECTIVE: The role of platelet derived serotonin (5-HT) in collagen induced human platelet aggregation was examined both in hirudinised plasma and whole blood. Hirudin was used to maintain normocalcaemia, thereby avoiding erroneous findings often obtained with citrated blood. METHODS: Platelet aggregation, aggregate growth, and primary platelet aggregation were quantified by optical aggregometry and platelet counting respectively. RESULTS: In plasma, platelet aggregation induced by 5-HT (3 microM) in the presence of adrenaline (1 microM) was inhibited by more than 85% by ICI 170809 (3 microM, IC50 0.1 microM), a specific platelet 5-HT2 antagonist. With collagen (0.5 micrograms.ml-1), ICI 170809 retarded the rate of aggregation by 30%, whereas aspirin abolished the response. In contrast, ICI 170809 inhibited collagen induced primary aggregation by less than 10% in both plasma and whole blood. CONCLUSIONS: (1) 5-HT contributes to collagen induced aggregate growth and 5-HT2 receptor antagonism with ICI 170809 retards the rate of growth. (2) This could explain the efficacy of 5-HT2 antagonists in limiting coronary thrombosis despite the limited role of 5-HT in primary aggregation.

The kinetics of platelet and fibrin deposition on to damaged rabbit carotid arteries in vivo: involvement of platelets in the initial deposition of fibrin.

Thrombus formation in the rabbit carotid artery has been studied kinetically in vivo using a minimally invasive technique utilising radioisotopes. Clamping of the carotid artery for 5 min resulted in the simultaneous accumulation of platelets and fibrin at the site of injury over the next 45 min. Under the electron microscope the response was seen to range from platelet monolayer adhesion to mural thrombus formation with fibrin deposition. In animals rendered thrombocytopenic, fibrin deposition was impaired during the first 15-20 minutes after injury. Basic coagulation times and fibrinogen concentration were within normal limits. In addition the injured vessels in these animals accumulated more radiolabelled albumin, but not erythrocytes, than injured vessels in control animals. The results may imply a role for platelets in the enhancement of fibrin deposition during the early part of the response to injury and in contributing to the maintenance of normal permeability following vessel injury.

Effect of dazoxiben, a thromboxane synthetase inhibitor on skin-blood flow following cold challenge in patients with Raynaud's phenomenon.

The effects of dazoxiben on finger-blood flow in response to cold challenge were studied in normal subjects and patients with Raynaud's phenomenon. In normal subjects concentrations of TXB2 and 6-oxo-PGF1 alpha were measured in blood taken from dorsal hand veins following cold challenge. In a parallel multicentre study we examined the effects of dazoxiben on finger temperature and capillary blood cell velocity in patients with Raynaud's phenomenon. Dazoxiben did not affect finger arterial inflow at rest or during cold challenge in patients or controls. However in both groups, recovery was quicker after cold challenge on dazoxiben treatment. In patients median flow was 5 ml (100(-1) ml) min-1 (range 1-10) v. 2 (0.5-15), P less than 0.05 dazoxiben v. placebo at 15 min after cold challenge. However, in normal subjects this did not prove to be statistically significant. In normal subjects there was a fall in TXB2 concentrations and relative rise in 6-oxo-PGF1 alpha following dazoxiben treatment indicating redirection of prostaglandin endoperoxides towards synthesis of PGI2. Comparison of the sum-total output of each eicosanoid following treatment with dazoxiben revealed a 65% reduction in TXB2 concentrations (P less than 0.025 compared with placebo) and a 40% increase in 6-oxo-PGF1 alpha concentrations (P less than 0.05 compared with placebo). However a simultaneous increase in concentrations of FPA indicated generation of thrombin, probably at the needle tip. Long-term treatment with dazoxiben resulted in no significant change in finger-skin temperature or capillary blood cell velocity, duration, or severity of attacks of Raynaud's phenomenon.